Manipulating cells, controlling their environment, and promoting conditions that mimic or illicit in vivo or natural cellular or tissue responses is an area of intense research. In the area of stem cells and regenerative medicine there is a particular need for methods and materials that replicate the native conditions where cells grow in vivo. Conditions that cells experience when removed from their native environment promote homeostasis, where cells change to adapt to their new environment, thus inducing cellular changes. Many of these processes are not elastic or reversible, therefore, cells cannot return to their native state. There is a strong need for materials and methods that promote natural cellular environments and minimize or control adverse cellular changes before cell and tissue engineering can reach its full potential.
Currently, materials are being developed that can support three-dimensional (3D) cell culturing conditions. Most of the work in 3D cell culture techniques involves either rotation of the flasks, the use of an exterior scaffold to which the cells can adhere, the use a magnetic fields to suspend cells, or some combination of these approaches.
For example, Felder in US2005054101, WO2005010162 describes a hydrogel substrate that forms an exterior scaffolding in which cells can grow and be supported in a 3D environment. This introduces an artificial substrate with which cells interact, rather than rapidly promoting cell-cell interactions, and although an improvement over 2D culturing, the scaffolding is likely to perturb the cells and remains in the finished product. Further, cells can grow on or in the microcarriers, but cells cannot be levitated in a manner where all around cell-cell contact/interaction is possible.
Nationally, there is a significant level of complexity involved in the fabrication of the microcarriers of Felder, which includes laborious chemistry and the need for complex equipment. Further, algimatrix, one of the main reagents in making the microcarriers, can be a source of endotoxins. Buoyancy control also seems to be relevant to facilitate levitation, and is controlled by the infusion of glass bubbles into the microcarriers, again contributing to complexity and difficulty. Finally, specialized hardware is required for agitation, which is needed achieve gas exchange and to prevent clumping of the microcarriers, and impellers are often used to agitate cells. However, the shear stress resulting from agitation is known to cause cell damage. Furthermore, agitation impairs any magnetic field shape control of 3D cultures.
Becker in US2009137018, WO2005003332 uses a coating of bioattractive magnetized core particles, thereby initiating adherence of the biological cells to the magnetized core particles and allowing their suspension in a magnetic field. The coating remains with the cells during culture, thus introducing an unnatural element in the culture and probably perturbing the cells. The inventors contemplate the use of a biodegradable coating that could eventually be eliminated, but none are disclosed, so it is not known if this approach would be successful. Furthermore, because cells are grown on the core of the microcarriers, the levitation of individual cells in which they can be brought together by magnetic levitation for the purpose of promoting cell-cell interaction is unlikely to take place. Therefore, it is not obvious that the rapid (hours) assembly of 3D multicellular structures due to cell-cell contact can be demonstrated when using microcarriers. Also, by growing cells on the microcarriers, the co-culture of different cells types, especially by levitating individual cells and then bringing them together magnetically, is not demonstrated. Finally, this system is cumbersome and not suitable for scale-up and high-throughput applications.
A better approach might be to temporarily magnetize cells, allowing for their 3D culture. For example, Akira in US2006063252, WO2004083412, WO2004083416 uses magnetic cationic liposomes (MCL) to magnetize cells by uptake of the liposomes. The magnetized cells are then grown in a sheet on the bottom of a plate using magnetic attraction, and then released for use. However, although able to produce sheets of cells, the cells are still grown on the bottom of a plate, and thus this is not true 3D culturing by magnetic levitation. Further, Shimizu and Akira et al. in a recent publication entitled “Effective Cell-Seeding Technique Using Magnetite Nanoparticles and Magnetic Force onto Decellularized Blood Vessels for Vascular Tissue Engineering” use magnetic guidance to seed cells onto a decellularized blood vessel.1 Their study shows encouraging results, but they do not use the magnetized cells as the source of tissue to be decellularized. The magnetized cells are only used to recellularize the decellularized blood vessels.
In patent application WO2010036957 by Souza, cells are levitated in a magnetic field by contacting the cells with a “hydrogel” comprising a bacteriophage with nanoparticles that are responsive to a magnetic field. In particular, filamentous phage, such as fd, fl, or M13 bacteriophage, are used. How the method works is not completely clear, but it is theorized that the phage provide a gel-like structure or assembly that coats the cells, and somehow assists the cells to uptake or adsorb the magnetically responsive nanoparticles. Thus, even when the hydrogel is washed away, the cells remains magnetically responsive, and can be levitated in an appropriate magnetic field. However, although the hydrogel is mostly washed away, the potential for phage infectivity or transfer of genetic material remains, and thus it is desired to provide a material that allows cell uptake or adsorption without the use of phage.
The present disclosure overcomes the shortcomings existing in the art by providing improved materials and methods that promote native cellular environments. These include utilizing compositions and methods for generating nanoparticle-based materials and preparing cells to enable 3D cell culturing, cell patterning, and cell imaging.